We will prepare 1-butanol extracts of fresh human melanoma cells to use as an allogeneic "vaccine" for active specific immunotherapy. The melanoma-associated antigens in each extract will be detected by a panel of mouse and human monoclonal antibodies and quantitated by a binding inhibition procedure we have developed. Human monoclonal antibodies will be continually developed from human-mouse and human-human fusions, with regional lymph node cells from melanoma patients as one partner and a nonsecretory mouse (M5) myeloma or a human B or myeloma cell line as the other partner. These human monoclonal antibodies will enable us to determine the presence of epitopes recognized by patients as "foreign", in the butanol extracts used as immunogens. The toxicity and immunological effects of a standard pool of 3 antigenic extracts will be tested in a formal Phase I trial, with metastatic melanoma patients. Two-fold escalations will be performed in each group for a total of 3 levels of dosage, with and without low-dose cyclophosphamide as a potentiator of immunity. Besides noting standard toxicity, we will also measure suppressor T cells by various complementary methods, to determine the possible occurence of this potentially harmful consequence of immunization. Several tests for antibodies to melanoma including immune adherence, protein A mixed hemadsorption and two tests for cytophilic antibodies (LAI and ADCC), will be performed. We have also developed a new titration method based on competitive inhibition of monoclonal antibody binding, with a sensitivity of 1-2 ng of antibody, to measure most sensitively the serum antibody response in the patients. Delayed hypersensitivity will be studied by skin testing with the pool of melanoma extracts. We will also explore limiting dilution analysis of cultivated T cells as a new means of detecting cytolytic antimelanoma T cells, since other tests of cell-mediated immunity have been inconsistently reliable. The developmental aspects of this proposal, with improvement of competitive binding, limiting dilution analysis and suppressor cell methods, in fact constitute an important rationale for the entire study. This closely monitored clinical trial may help to define the most important antigens to include in a "vaccine", leading to their selective use in future studies of specific active immunotherapy for melanoma.